Long-acting microbial colonization unit for remediation of heavy metals in soil and preparation method and application thereof

Abstract

This invention discloses a long-lasting microbial colonization unit for heavy metal remediation in soil, its preparation method, and its application, belonging to the field of soil remediation technology. The long-lasting microbial colonization unit has a core-shell structure: the core is an organic-inorganic-biological coupled composite material formed by gradient coupling co-aging of an organic matrix, modified hydrotalcite, and a microbial nutrient inducer; the shell is a composite coating layer of sodium alginate-carboxymethyl chitosan-nano-hydroxyapatite surrounding the core. The organic matrix is ​​prepared by composting bacterial residue and chicken manure with a composite bacterial agent containing Bacillus licheniformis, Bacillus subtilis, and Sinapis algae-degrading bacteria. This invention, through the synergistic effects of introducing saline-alkali source strains, gradient coupling co-aging process, and composite coating layer, achieves efficient colonization and long-term survival of microorganisms in contaminated soil, simultaneously completing heavy metal passivation and achieving a remediation time of ≥120 days, making it particularly suitable for the remediation of moderately to severely contaminated soils.

Description

Technical Field

[0001] This invention belongs to the field of soil remediation technology, specifically relating to a long-lasting microbial colonization unit for heavy metal remediation in soil, its preparation method, and its application. Background Technology

[0002] Heavy metal pollution in arable land and greenhouse soils poses a serious threat to agricultural product safety. Moderately to severely polluted soils are often accompanied by complex adverse conditions such as salinization and infertility, making remediation even more difficult. Traditional soil remediation technologies have limitations such as high cost and the potential for secondary pollution. Existing technologies include bioremediation using microorganisms (such as Bacillus) or the adsorption and passivation of heavy metals using materials such as biochar and clay minerals. However, these methods face the following technical bottlenecks: (1) When microbial agents are applied directly, their survival rate in the soil environment is low and their effective period is short. It is difficult for them to effectively colonize in the soil and form a dominant microbial community. This is the core problem that restricts the application of microbial remediation technology. (2) Powdered or granular inorganic adsorbent materials (such as clay minerals, hydrotalcite, etc.) are easily washed away by rainwater during use, resulting in insufficient durability of the repair effect; (3) Organic-inorganic composite materials are mostly simple physical mixtures, and the components are easily separated in the soil, making it impossible to form a synergistic remediation system; (4) Existing remediation materials are mostly designed for a single function, making it difficult to integrate and control the release of multiple functions such as heavy metal passivation, microbial colonization, and soil improvement; (5) Conventional microbial agents are poorly adapted to complex soils with moderate to severe pollution and accompanied by saline-alkali stress, and are difficult to play a remediation role.

[0003] To address the aforementioned problems, this invention proposes a novel technical concept: combining *Sinapis algae-degrading* bacteria isolated from saline-alkali soil with *Bacillus licheniformis* and *Bacillus subtilis* to construct a multifunctional microbial community with tolerance to complex stresses. Simultaneously, functional microorganisms, nutrient substrates, and inorganic adsorbent materials are deeply integrated and structurally designed at the microscale through a specific process, forming a functional colonization unit capable of providing survival, reproduction, and long-term nutrient supply for the microorganisms. This unit, through a unique gradient-coupled co-aging process and a functional composite coating layer design, achieves efficient colonization and long-term survival of microorganisms in contaminated soils (including saline-alkali stress soils), while also endowing the materials with gradient slow-release properties and multiple remediation functions, fundamentally overcoming the shortcomings of existing technologies. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies, such as difficulty in microbial colonization, short remediation cycle, easy separation of components, lack of functional synergy, and poor adaptability to adversity. It provides a long-lasting microbial colonization unit and its preparation method that has strong component binding, gradient slow-release characteristics, can achieve efficient microbial colonization and long-term passivation of heavy metals, and is especially suitable for the remediation of moderately to severely polluted soils.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A long-lasting microbial colonization unit for soil heavy metal remediation is characterized by having a core-shell structure, comprising a core and a shell. The core is an organic-inorganic-biological coupled composite material formed by gradient coupling co-aging of an organic matrix, modified hydrotalcite, and a microbial nutrient inducer. The shell is a sodium alginate-carboxymethyl chitosan-nanohydroxyapatite composite coating layer wrapped around the core. The organic substrate is prepared by fermenting and composting bacterial residue and chicken manure with a compound bacterial agent containing Bacillus licheniformis, Bacillus subtilis, and Sinica microorganisms that decompose colloids.

[0006] Furthermore, the *Bacillus licheniformis* was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 27, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35039; *Bacillus subtilis* was deposited at the same center on November 25, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36767; and the strain of *Sinopterinaria colloidis*, with strain number CGMCC No. 1.11000, was purchased from the CGMCC.

[0007] Furthermore, the mass ratio of the organic matrix, modified hydrotalcite, and microbial inducer in the core is (70-80):(15-25):(1-5); the modified hydrotalcite is a magnesium-aluminum-iron ternary hydrotalcite with the chemical composition [Mg3Al2O3]. 1-x Fe x [(OH)8]2(CO3)·nH2O, where x=0.05-0.15 and the particle size is 150-200 mesh.

[0008] Furthermore, a co-precipitation method was used to prepare magnesium-aluminum-iron ternary hydrotalcite.

[0009] Furthermore, the microbial nutrient inducer is a complex of yeast extract and trehalose in a mass ratio of 2:1.

[0010] Furthermore, the method for preparing the organic substrate is as follows: the bacterial residue is mixed with chicken manure, and a compound bacterial agent containing Bacillus licheniformis, Bacillus subtilis and Sinica microorganisms that decompose colloids is inoculated. The mixture is then aerobically fermented at 55-60℃ for 45-50 days until the seed germination index is greater than 80%, thus obtaining a decomposed organic substrate.

[0011] Furthermore, the dry weight ratio of the bacterial residue to chicken manure was (70-75):(25-30), and the viable counts of Bacillus licheniformis, Bacillus subtilis, and Sinapis algae-degrading bacteria were mixed in a 1:1:1 ratio, resulting in a total viable count of 2×10⁻⁶. 8 CFU / mL, with an inoculum size of 0.2%-0.4% of the total mass of the mixture.

[0012] Furthermore, the gradient coupling co-aging process includes the following steps: (1) Mix the organic matrix, modified hydrotalcite and microbial nutrient inducer, and adjust the moisture content to 45-55%; (2) Under hypoxic conditions, periodic temperature variation aging is carried out at 15-35℃, with each cycle lasting 48 hours and aging for 5-7 days; (3) Transfer to aerobic conditions and continue aging at room temperature for 10-14 days, turning the pile over every 3 days during this period.

[0013] Furthermore, the oxygen deficiency condition is defined as CO2 gas concentration ≥ 5% and oxygen concentration < 5%.

[0014] Furthermore, the high-temperature period (30-35℃) is maintained for 12 hours, and the low-temperature period (15-20℃) is maintained for 12 hours.

[0015] A method for preparing a long-lasting microbial colonization unit for soil heavy metal remediation includes the following steps: Organic matrix and modified hydrotalcite were prepared separately. The organic matrix, modified hydrotalcite and microbial nutrient inducer were mixed in proportion and subjected to gradient coupling co-aging treatment. The co-aged material was granulated and dried to obtain wet microspheres. The microspheres were coated with sodium alginate-carboxymethyl chitosan-nano hydroxyapatite composite coating solution, then cured with cross-linking curing solution, and dried to obtain long-term microbial colonization unit for soil heavy metal remediation.

[0016] Furthermore, the composite coating solution contains 2% sodium alginate, 1% carboxymethyl chitosan, and 0.5% nano-hydroxyapatite; the cross-linking curing solution contains 5% calcium chloride and 0.5% boric acid.

[0017] Furthermore, the degree of substitution of carboxymethyl chitosan is ≥80%, and the particle size of nano-hydroxyapatite is 50-100nm.

[0018] Furthermore, the weight gain rate of ovule coating is 15-20%.

[0019] The present invention also provides the application of the long-lasting microbial colonization unit in the remediation of heavy metal contaminated soil.

[0020] Furthermore, depending on the degree of soil pollution, the dosage is 1-3 tons per acre, and the method of application is to apply it to the root zone or mix it with the topsoil before crop transplanting.

[0021] Compared with the prior art, the present invention has the following significant advantages: (1) The concept of “microbial colonization unit” is proposed: Unlike the existing technology that simply regards remediation materials as adsorbents or carriers, this invention starts from the perspective of microbial ecology and designs the material as a functional unit that can actively promote the survival and reproduction of functional microorganisms in polluted soil, thus solving the core problems of difficult microbial colonization and low survival rate.

[0022] (2) Introducing saline-alkali source functional strains to significantly enhance stress adaptability: The *Sinapis algae-degrading* strain used in this invention was isolated from saline-alkali soil and has natural saline-alkali tolerance and stress adaptability. When combined with *Bacillus licheniformis* and *Bacillus subtilis*, a multifunctional bacterial community with complex stress tolerance was constructed. This community exhibited excellent survival rate and colonization ability in heavy metal-saline-alkali soil, a characteristic not possessed by conventional strains.

[0023] (3) Synergistic effect of functional microbial community: The three-strain compound system has complementary functions—Bacillus licheniformis has strong salt tolerance and adapts to harsh environments; Bacillus subtilis produces abundant enzymes and promotes the decomposition of organic matter; and Sinica microorganism, which decomposes colloids, originates from a saline-alkali environment and not only has the ability to decompose silicate minerals, but also carries unique stress-adaptation genes, which can maintain metabolic activity under high salt and high pH conditions. The three work together to have multiple functions such as heavy metal passivation, soil improvement, crop growth promotion, and stress adaptation.

[0024] (4) Optimization of hydrotalcite structure to enhance adsorption selectivity: The magnesium-aluminum-iron ternary hydrotalcite is adopted. The introduction of iron not only enhances the adsorption selectivity of heavy metals such as Cd, Pb and As, but also gives the material weak magnetism, which is convenient for later monitoring and potential recovery. At the same time, iron ions can promote electron transfer in microorganisms and enhance microbial activity.

[0025] (5) Innovative Gradient Co-aging Process: Through a gradient coupling co-aging process of "alternating anoxic / aerobic conditions + temperature-induced degradation," organic matter, hydrotalcite, and nutrient inducers are chemically bonded and physically coated at the microscopic level, creating a stable composite structure. The anoxic stage promotes anaerobic fermentation to produce organic acids, activating the surface of the hydrotalcite; the aerobic stage enhances the humification of organic matter and the reproduction of microorganisms. This process upgrades the components from physical mixing to chemical coupling, significantly improving the structural stability and functional synergy of the composite.

[0026] (6) Environmental Response of Functional Composite Coating Layer: Carboxymethyl chitosan and nano-hydroxyapatite are introduced. Carboxymethyl chitosan has pH-responsive swelling properties, which can moderately swell and promote release in acidic soil environments, and remain stable in neutral environments. Nano-hydroxyapatite not only enhances the adsorption capacity of heavy metals, but also slowly releases calcium and phosphorus nutrients and promotes microbial reproduction. The coating layer realizes intelligent regulation and gradient slow release of the core components.

[0027] (7) Continuous activation by nutrient inducers: The yeast extract and trehalose compound inducer added to the core can be slowly released under soil moisture stimulation, continuously activating the dormant functional microorganisms in the core, enabling them to quickly recover and reproduce under suitable conditions, and significantly extending the repair cycle.

[0028] (8) Long-term synergistic remediation, especially suitable for complex stress soils: This product can achieve a continuous remediation effect of ≥120 days in the soil, simultaneously achieving heavy metal passivation, soil structure improvement, microbial community activation and crop growth promotion. It is especially suitable for the treatment and ecological restoration of moderate to severe complex pollution (such as heavy metal-salt-alkali complex) soils. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0030] Example 1 A method for preparing a long-lasting microbial colonization unit for soil heavy metal remediation includes the following steps: (1) Preparation of organic matrix Take 45% moisture content oyster mushroom residue (crushed to less than 2cm) and mix it with dry chicken manure at a dry basis weight ratio of 72:28. Take 1000 kg of the mixture and inoculate it with a compound bacterial solution composed of Bacillus licheniformis (CGMCC No. 35039), Bacillus subtilis (CGMCC No. 1.1086), and Microsporum desmolyticus (CGMCC No. 1.11000) at a viable count ratio of 1:1:1 (total viable count 2×10⁻⁶). 83.0 kg (0.3%) of material (CFU / mL) was used. The moisture content of the material was adjusted to 52% using a solution containing 0.3% potassium dihydrogen phosphate and 0.08% magnesium sulfate. Aerobic fermentation was carried out in a pile, with the high-temperature period (>55℃) controlled for 15 days, the average pile temperature being 58℃, and the total fermentation cycle being 48 days. The seed germination index (GI) of the material at the end of fermentation was 86%. After that, it was aged for 7 days in a rain-protected environment to obtain an organic substrate with a viable count ≥5×10⁻⁶. 8 CFU / g; (2) Preparation of modified hydrotalcite Prepare a mixed salt solution containing Mg(NO3)2·6H2O, Al(NO3)3·9H2O, and Fe(NO3)3·9H2O, with a Mg:Al:Fe molar ratio of 3:0.9:0.1 and a total metal ion concentration of 1.0 mol / L. Also prepare a mixed alkaline solution containing NaOH and Na2CO3. - [CO3] = 2.5 mol / L 2- [Mg3Al] = 0.5 mol / L. The two solutions were added dropwise to the reactor in parallel streams with stirring, controlling the pH at 10 ± 0.2 and the temperature at 80℃. After the addition was complete, stirring was continued for 24 hours for crystallization. The product was filtered, washed until neutral, dried at 80℃ for 12 hours, and ground through a 200-mesh sieve to obtain [Mg3Al]. 0.9 Fe 0.1 [(OH)8]2(CO3)·4H2O, thus obtaining modified hydrotalcite.

[0031] (3) Preparation of microbial nutrient inducers Yeast extract and trehalose were mixed evenly at a mass ratio of 2:1 to obtain a microbial nutrient inducer.

[0032] (4) Gradient-coupled co-aging treatment Take 750 kg of the organic matrix (dry basis) obtained in step (1), 210 kg of the modified hydrotalcite obtained in step (2), and 40 kg of the nutrient inducer obtained in step (3), mix them evenly in a mixer, and adjust the moisture content to 50%. Transfer the mixture to a sealed aging container, introduce CO2 gas to a concentration ≥5% and oxygen concentration <5%, and age it for 6 days in a cyclic temperature change environment of 15-35℃ (one cycle every 48 hours, with a high temperature period of 30-35℃ for 12 hours and a low temperature period of 15-20℃ for 12 hours). During this period, take samples every 2 days to test pH and microbial activity.

[0033] After the anaerobic aging process, the material is transferred to a well-ventilated and shady place, spread out to a thickness of 30 cm, covered with a breathable shade net, and subjected to aerobic aging for 12 days. During this period, the material is turned over every 3 days with a turning machine to keep it loose and breathable. After the aerobic aging process, the material is dark brown, has a soil-like aroma, and the organic matter-hydrotalcite complex structure is stable.

[0034] (5) Granulation and drying The co-aged material was granulated into wet pellets with a diameter of approximately 4.0 mm using a twin-screw extruder. The wet pellets were then dried in hot air circulation at 40°C until the moisture content reached 32%, yielding micro-wet pellets.

[0035] (6) Composite coating and cross-linking curing Preparation of coating solution: Weigh 20g of sodium alginate and dissolve it in 1000 mL of deionized water, stirring until completely dissolved; add 10g of carboxymethyl chitosan (85% substitution degree) and stir to dissolve; add 5g of nano-hydroxyapatite (80 nm particle size) and disperse at high speed for 30 minutes to obtain composite coating solution.

[0036] In a fluidized bed coating machine, micro-wet beads are preheated to 35°C and coated by bottom spraying. The inlet air temperature is 45°C, and the atomization pressure is 0.2 MPa, resulting in a coating weight gain of approximately 18%. After coating, a crosslinking and curing solution containing 5% calcium chloride and 0.5% boric acid is prepared and sprayed into the fluidized bed for crosslinking and curing for 10 minutes. Finally, the solution is dried at 42°C until the moisture content is ≤25%, yielding a long-lasting microbial colonization unit.

[0037] Product performance characterization: The performance of the finished product was tested, and the results are as follows: Physical properties: average particle size 4.3 mm; after soaking in soil simulation solution (prepared according to GB / T 32740-2016) for 15 days, the swelling rate is 28%; measured by a texture analyzer, it can withstand 76% compressive strain and the rupture compressive stress is 102 kPa.

[0038] Bioactivity: The viable bacterial count inside the fragmented microsphere core was determined using the plate count method to be 4.2 × 10⁻⁶. 8 CFU / vial. Microbial community structure analysis showed that the ratio of the three strains remained at approximately 1:0.9:0.8, indicating a stable microbial community structure.

[0039] Heavy metal adsorption performance: in Cd-containing 2+ (10 mg / L) and Pb 2+ Add 1 g / L of colonization unit to a mixed solution of (50 mg / L), shake for 24 hours, and then apply to Cd. 2+ and Pb 2+ The adsorption rates were 95.1% and 92.3%, respectively.

[0040] Slow-release performance: After continuous immersion in soil slurry for 120 days, the number of viable bacteria in the core remains ≥10. 7 CFU / capsule, with a cumulative release rate of 78% of the nutrient inducer, exhibiting a gradient slow-release characteristic.

[0041] Comparative Example 1 Compared with Example 1, this comparative example is identical to Example 1 except that gradient coupling co-aging treatment is not performed, and the organic matrix, modified hydrotalcite, and microbial nutrient inducer are physically stirred in a high-speed mixer for only 30 minutes and then granulated and coated.

[0042] Comparative Example 2 Compared with Example 1, this comparative example does not perform the composite coating and cross-linking curing steps. The micro-wet granules are directly dried to obtain the final product. The remaining steps and raw materials are the same as in Example 1.

[0043] Comparative Example 3 This comparative example uses the same organic matrix, modified hydrotalcite, and microbial nutrient inducer as Example 1. No co-aging or granulation is performed; instead, the organic matrix, modified hydrotalcite, and microbial nutrient inducer are simply physically mixed at a dry matrix mass ratio of 750:510:40 and applied to the soil in powder form.

[0044] Comparative Example 4 Compared with Example 1, this comparative example uses the same raw materials and steps as Example 1, except that the composite coating liquid is a conventional coating (2% sodium alginate-2% bentonite composite liquid, and the crosslinking curing agent is 4% calcium chloride).

[0045] Comparative Example 5 Compared with Example 1, this comparative example differs from Example 1 except that in the organic matrix preparation process, the compound bacterial agent is simply a 1:1 mixture of Bacillus licheniformis and Bacillus subtilis with live bacteria, and no *Sinocyclohexane* is added. All other raw materials and steps are the same as in Example 1.

[0046] Comparative Example 6 Compared with Example 1, the comparative example replaced the gelatinous Bacillus thymosus (CGMCC No.1.11000) with "Mizutani Shin" gelatinous Bacillus purchased online, while the other raw materials and steps were the same as in Example 1.

[0047] Performance testing Verification experiment on the effects of ordinary contaminated soil (1) Experimental design A contaminated soil sample was taken from a farmland (pH=6.5, DTPA extractable Cd=2.10mg / kg, Pb=142mg / kg, total salt content 0.25%). The soil was air-dried, sieved, and then divided into 5kg plastic basins.

[0048] Set up the following 8 treatment groups, with 3 replicates in each group: CK: Blank control, no repair materials added; T1 (Example 1): Add 2% (w / w, equivalent to 3 tons / acre based on approximately 150 tons of topsoil weight per acre) of the long-lasting microbial colonization unit of this invention. CP1 (Comparative Example 1): Add 2% non-co-aged microspheres CP2 (Comparative Example 2): Add 2% uncoated embryo beads CP3 (Comparative Example 3): A simple mixture of organic matrix, modified hydrotalcite, and nutrient inducer in the same amount as T1. CP4 (Comparative Example 4): 2% conventional coated microspheres (sodium alginate-bentonite coating) were added. CP5 (Comparative Example 5): Added 2% of non-degradable gelatinous Chinese microbeads CP6 (Comparative Example 6): Added 2% of conventional "Mizutani Shin" Bacillus subtilis microspheres All materials were thoroughly mixed with soil, and the soil moisture content was maintained at 60%-70% of field capacity. The mixture was then incubated at room temperature. Samples were taken at 30, 90, and 150 days of incubation to analyze the effective heavy metal removal rate. The effect of each treatment group on the number of soil microorganisms at 150 days was also statistically analyzed. The statistical results are shown in Tables 1 and 2.

[0049] Table 1. Removal effect of each treatment group on available heavy metals in soil Table 2. Effects of each treatment group on soil microbial abundance Experimental Verification of the Effects of Soil Pollution from Salt-Alkali Compound Pollution Experimental Design Contaminated soil from a saline-alkali farmland (pH=8.3, EC=1.8 mS / cm, total salt content 0.42%, DTPA extractable Cd=1.85 mg / kg, Pb=98 mg / kg) was collected. The soil was air-dried, sieved, and then divided into plastic basins, with 5 kg of soil in each basin.

[0050] Key treatment groups were selected for comparison: T1 (Example 1), CP5 (non-degrading Bacillus subtilis), CP6 (conventional Bacillus subtilis), and CK (blank control), all with an addition amount of 2% (w / w). The culture conditions were the same as those in the above-mentioned efficacy verification experiment on ordinary contaminated soil. Samples were taken and analyzed at 30, 90, and 150 days. The statistical results are shown in Tables 3 and 4.

[0051] Table 3. Removal effect of each treatment group on available heavy metals in saline-alkali soil. Table 4. Effects of each treatment group on microbial abundance in saline-alkali soil at 150 days. The following conclusions can be drawn from the experimental results in Table 1-4: The long-term remediation effect is significant. In ordinary contaminated soil, the T1 group of this invention still maintains a Cd removal rate of 73.5% after 150 days, which is significantly better than all comparative groups. In saline-alkali compound contaminated soil, the Cd removal rate of the T1 group reached 68.2% after 150 days, while the CP5 group was only 38.6% and the CP6 group was 45.3%, proving that the present invention can still maintain excellent remediation effect in adverse soil. The saline-alkali source strain has obvious advantages in adverse adaptation. Comparing the performance of T1 and CP6 in saline-alkali soil, the Cd removal rate (68.2%) and Pb removal rate (62.5%) of T1 after 150 days are significantly better than those of CP6 (Cd 45.3%, Pb 39.5%), and the colonization rate of functional bacteria (58%) is significantly better than that of CP6 (26%), proving that the *Sinapis algae-degrading* strain derived from saline-alkali soil has a natural advantage in adverse adaptation.

[0052] The microbial colonization effect is outstanding. In ordinary soil, the colonization rate of functional bacteria in group T1 reaches 71%, and in saline-alkali soil it still reaches 58%, while group CP5 is only 12% and group CP6 is 26%. This proves that the microbial colonization unit design of the present invention has successfully achieved efficient colonization and long-term survival of functional microorganisms in polluted soil (including stress soil). Synergistic effects of the components are indispensable. Firstly, comparing T1 with CP4 (conventional coating), CP5 (lacking *Sinapis algae-degrading*), and CP6, T1 showed significantly better remediation effects and microbial colonization rates at all time points. This demonstrates the importance of the synergistic effect between the composite coating layer and the saline-alkali source functional strains of this invention. The unique contribution of the saline-alkali source strains, particularly the introduction of *Sinapis algae-degrading*, not only enhances the overall stress adaptability of the microbial community but also endows the colonization units with excellent performance in saline-alkali compound-contaminated soils—something that conventional strain combinations cannot achieve. Secondly, the co-aging treatment process is necessary. Compared with CP1 and CP3, T1 showed significantly better performance, demonstrating that the gradient coupling co-aging process enabled deep integration of the components at the microscopic level, forming a stable composite structure and functional synergy that cannot be achieved by simple mixing. Regarding the functional advantages of the coating layer, compared with CP4, T1's 150-day repair effect (73.5%) was significantly better than CP4's (53.7%), proving that the carboxymethyl chitosan-nano hydroxyapatite composite coating layer has better environmentally responsive sustained-release performance and functional enhancement effect compared to conventional sodium alginate-bentonite coating.

[0053] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A long-lasting microbial colonization unit for soil heavy metal remediation, characterized in that, It has a core-shell structure, including a core and a shell. The core is an organic-inorganic-biological coupled composite material formed by gradient coupling co-aging of an organic matrix, modified hydrotalcite, and microbial nutrient inducer. The shell is a composite coating layer of sodium alginate-carboxymethyl chitosan-nano hydroxyapatite wrapped around the core; The organic substrate is composed of bacterial residue and chicken manure containing Bacillus licheniformis. Bacillus licheniformis Bacillus subtilis Bacillus subtilis It is prepared by fermentation and composting of a compound inoculant of *Sinomyces chinensis* that decomposes colloids.

2. The long-acting microbial colonization unit for soil heavy metal remediation according to claim 1, characterized in that, The Bacillus licheniformis Bacillus licheniformis Bacillus subtilis was deposited on June 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35039. Bacillus subtilis The sample was deposited on November 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36767; the strain number of *Sinopterinella spp.* was CGMCC No. 1.11000, purchased from the China General Microbiological Culture Collection Center.

3. The long-lasting microbial colonization unit for soil heavy metal remediation according to claim 1, characterized in that, The core contains an organic matrix, modified hydrotalcite, and a microbial inducer in a mass ratio of (70-80):(15-25):(1-5); the modified hydrotalcite is a magnesium-aluminum-iron ternary hydrotalcite with the chemical composition [Mg3Al2O3]. 1-x Fe x [(OH)8]2(CO3)·nH2O, where x=0.05-0.15 and the particle size is 150-200 mesh.

4. The long-lasting microbial colonization unit for soil heavy metal remediation according to claim 1, characterized in that, The microbial nutrient inducer is a complex of yeast extract and trehalose in a mass ratio of 2:

1.

5. The long-lasting microbial colonization unit for soil heavy metal remediation according to claim 1, characterized in that, The method for preparing the organic substrate is as follows: mix the bacterial residue with chicken manure, inoculate with a compound bacterial agent containing Bacillus licheniformis, Bacillus subtilis and Sinica microorganisms that decompose colloids, and ferment aerobicly at 55-60℃ for 45-50 days until the seed germination index is greater than 80%, to obtain a decomposed organic substrate.

6. The long-lasting microbial colonization unit for soil heavy metal remediation according to claim 5, characterized in that, The dry weight ratio of the mushroom residue to chicken manure is (70-75):(25-30). Bacillus licheniformis, Bacillus subtilis, and Sinapis algae-degrading bacteria are mixed at a viable count ratio of 1:1:1, resulting in a total viable count of 2×10⁻⁶. 8 CFU / mL, with an inoculum size of 0.2%-0.4% of the total mass of the mixture.

7. The long-lasting microbial colonization unit for soil heavy metal remediation according to claim 1, characterized in that, The gradient coupling co-aging process includes the following steps: (1) Mix the organic matrix, modified hydrotalcite and microbial nutrient inducer, and adjust the moisture content to 45-55%; (2) Under hypoxic conditions, periodic temperature variation aging is carried out at 15-35℃, with each cycle lasting 48 hours and aging for 5-7 days; (3) Transfer to aerobic conditions and continue aging at room temperature for 10-14 days, turning the pile over every 3 days during this period.

8. A method for preparing the long-acting microbial colonization unit for soil heavy metal remediation according to any one of claims 1-7, characterized in that, Includes the following steps: Organic matrix and modified hydrotalcite were prepared separately; Organic matrix, modified hydrotalcite and microbial nutrient inducer were mixed in proportion and subjected to gradient coupling co-aging treatment; the co-aged material was granulated and dried to obtain wet microspheres; the microspheres were coated with sodium alginate-carboxymethyl chitosan-nano hydroxyapatite composite coating solution, then cured with cross-linking curing solution, and dried to obtain long-term microbial colonization unit for soil heavy metal remediation.

9. The long-lasting microbial colonization unit for soil heavy metal remediation according to claim 8, characterized in that, The composite coating solution contains 2% sodium alginate, 1% carboxymethyl chitosan, and 0.5% nano-hydroxyapatite; the cross-linking curing solution contains 5% calcium chloride and 0.5% boric acid.

10. The application of a long-lasting microbial colonization unit according to any one of claims 1-7 in the remediation of heavy metal contaminated soil.

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